When light is directed onto a molecule, the vast majority of the incident photons are elastically scattered without a change in frequency; however, the energy of some of the incident photons (approximately 1 in every 107 incident photons) is coupled into distinct vibrational modes of the molecule's bonds. Such coupling causes some of the incident light to be inelastically scattered by the molecule with a range of frequencies that differ from the range of the incident light. This is termed the Raman effect. By plotting the frequency of such inelastically scattered light against its intensity, the unique Raman spectrum of the molecule under observation is obtained. Analysis of the Raman spectrum of an unknown sample can yield information about the sample's molecular composition.
The incident illumination for Raman spectroscopy, usually provided by a laser, can be concentrated to a small spot if the spectroscope is built with the configuration of a microscope. Since the Raman signal scales linearly with laser power, light intensity at the sample can be very high in order to optimize sensitivity of the instrument. Moreover, because the Raman response of a molecule occurs essentially instantaneously (without any long-lived highly energetic intermediate states), photobleaching of the Raman-active molecule—even by this high intensity light—is impossible. This places Raman spectroscopy, in stark contrast to fluorescence spectroscopy, in which photobleaching dramatically limits many applications.
The Raman effect can be significantly enhanced by bringing the Raman-active molecule(s) close (<50 Å) to a nanometer-scale roughened metal surface. Bringing molecules in close proximity to metal surfaces is typically achieved through adsorption of the Raman-active molecule onto suitably roughened gold, silver, or copper or other free electron metals. Surface-enhancement of the Raman activity is also observed with metal colloidal particles, metal films on dielectric substrates, and metal particle arrays. The mechanism by which this surface-enhanced Raman scattering (SERS) occurs is not well understood, and is thought to result from a combination of (i) electromagnetic effects, surface plasmon resonances in the metal that enhance the local intensity of the light, and (ii) chemical effects, formation and subsequent transitions of charge-transfer complexes between the metal surface and the Raman-active molecule.
SERS allows detection of molecules attached to the surface of a single gold or silver nanoparticle. A Raman-enhancing metal that has associated or bound to it a Raman-active molecule(s) is referred to as a SERS-active nanoparticle. Such SERS-active nanoparticles can have utility as optical tags. For example, SERS-active nanoparticles can be used in immunoassays when conjugated to an antibody against a target molecule of interest. If the target of interest is immobilized on a solid support, then the interaction between a single target molecule and a single nanoparticle-bound antibody can be detected by searching for the Raman-active molecule's unique Raman spectrum. Furthermore, because a single Raman spectrum (from 100 to 3500 cm−1) can detect many different Raman-active molecules, different SERS-active nanoparticles can be used in multiplexed assay formats.
In U.S. patent application Ser. No. 09/680,782, filed Oct. 6, 2000, entitled “Surface Enhanced Spectroscopy-Active Composite Nanoparticles,” incorporated herein by reference in its entirety, and hereinafter referred to as the '782 application, SERS-based tags are described. Each SERS-active composite nanoparticle (SACN) consists of a SERS-active metal nanoparticle; a submonolayer, monolayer, or multilayer of SERS-active species in close proximity to the metal surface; and an encapsulating shell consisting of a polymer, glass, or other dielectric material. This places the SERS-active molecule (alternately referred to herein as the “analyte,” not to be confused with the species in solution that is ultimately being quantified) at the interface between the metal nanoparticle and the encapsulant. In other words, “analyte” as used in the specification is not the species that are tagged by the SACNs (which would be the conventional usage), but the one or more spectroscopy-active (e.g., Raman-active) molecules attached to or associated with the metal nanoparticles themselves. Because it is the signal from the SES-active composite nanoparticle that is being detected and measured, the Raman-active molecules of the composite nanoparticles themselves are referred to as the analyte. This special definition has been chosen by the applicant to underscore that it is the Raman spectrum of the Raman-active molecules, rather than the item tagged or labeled by the SACN, that is the source of the signal being measured.
The analyte molecule can be chosen to exhibit extremely simple Raman spectra, because there is no need for the species to absorb visible light. This, in turn, allows multiple SACN particles, each with different analyte molecules, to be fabricated such that the Raman spectra of each analyte can be distinguished in a mixture of different types of SACN particles.
SACNs are easily handled and stored. Because of the encapsulant, they are also aggregation resistant, stabilized against decomposition of the analyte in solvent and air, chemically inert, and easily centrifuged and redispersed without loss of SERS activity. Most importantly, the encapsulant shells of SACNs may be readily derivatized by standard techniques. This allows SACNs to be conjugated to molecules (including biomolecules such as proteins and nucleic acids) or to solid supports without interfering with the Raman activity of the SACNs. Unlike metal nanoparticles, SACNs can be evaporated to dryness, and then completely redispersed in solvent. Using the techniques provided in the '782 application, it is possible to fabricate SACNs that are individually detectable using SERS.
The SACNs provided by the '782 application are uniquely identifiable nanoparticles. They can be used in virtually any situation in which it is necessary to label molecules or objects (including beads and other types of solid support) with an optical tag. Biomolecules can be conjugated readily to the exterior of SACNs by standard techniques, thereby allowing the particles to function as optical tags in biological assays. SACNs can be used in virtually any assay that uses an optical tag such as a fluorescent label; however, as optical tags, SACNs have several distinct advantages over fluorescent labels. These advantages include vastly more sensitive detection, chemical uniformity, and the resistance of the SERS activity to photobleaching or photodegradation. A further benefit of using SACNs as optical tags is the ease with which individual SACNs having different SERS activities may be resolved from one another. At least twenty different SACNs are resolvable from one another using a simple Raman spectrometer. This enables multiplexed assays to be performed using a panel of different SACNs, each having a unique and distinguishable SERS activity.
U.S. Pat. No. 6,149,868, entitled “Surface Enhanced Raman Scattering From Metal Nanoparticle-Analyte-Noble Metal Substrate Sandwiches,” incorporated herein by reference in its entirety, and hereinafter referred to as the '868 patent, teaches that the Raman intensity of SERS-active molecules can be significantly enhanced by conjugating the molecule to colloidal metal nanoparticles, and then absorbing or covalently-attaching the metal nanoparticles to a macroscopic SERS substrate, such as an aggregated Ag sol or a roughened Ag electrode. In doing so, sandwiches are formed between the metal nanoparticle and the macroscopic SERS substrate, with the SERS-active molecule lying between the two metal surfaces. It is known that the enhancement in SERS-activity in this configuration results from large increases in the electric field between the colloidal metal nanoparticles and the macroscopic SERS substrate. Although the sandwiches of the '868 patent are themselves useful as SERS substrates, the macroscopic dimensions of the SERS-active substrate onto which the nanoparticles are absorbed preclude them from being optimal optical tags for biomolecular labeling. Moreover, the structural heterogeneity of the SERS-active substrates onto which the colloidal particles are absorbed means that a sandwich is not formed at every site where a colloidal particle associates with the SERS-active substrate.
Recently, SERS spectra have been observed for single molecules on the surface of colloidal metal nanoparticles, with enhancement factors of 1014-1015. Although the mechanisms for single-molecule and single-particle SERS are still unknown, it is believed that the large enhancement factors are obtained only at the interstitial sites between two particles or at locations outside sharp surface protrusions, so-called “hot spots.” In fact, it has been hypothesized that SERS spectra of large numbers of molecules are dominated by single molecules adsorbed at special surface sites. One recent study of SERS of rhodamine 6G molecules on the surface of silver nanoparticles found that SERS activity occurred only for clusters of at least two individual silver particles, and not for isolated particles (A. M. Michaels et al., “Ag Nanocrystal Junctions as the Site for Surface-Enhanced Raman Scattering of Single Rhodamine 6G Molecules,” J. Phys. Chem. B 2000, 104, 11965-11971). These clusters were not deliberately prepared, but rather were formed randomly by spin-casting a solution of R6G and colloidal silver onto a polylysine-coated quartz cover slip. In addition, there were no free clusters of particles formed; all of the clusters were formed on the surface of the cover slip.
Rod-shaped nanoparticles and methods for their use are described in detail in U.S. patent application Ser. No. 09/598,395, filed Jun. 20, 2000, and its continuation-in-part, U.S. patent application Ser. No. 09/677,198, filed Oct. 2, 2000, both entitled “Colloidal Rod Particles as Nanobar Codes,” and both incorporated herein by reference in their entirety. Also incorporated herein by reference in their entirety are U.S. patent application Ser. No. 09/677,203, entitled “Method of Manufacture of Colloidal Rod Particles as Nanobar Codes,” and U.S. patent application Ser. No. 09/676,890, “Methods of Imaging Colloidal Rod Particles as Nanobar Codes,” both filed Oct. 2, 2000. The latter application describes flow cytometry techniques to quantify fluorescent nanoparticles, optical microscopy fluorescence detection of nanoparticles, and TEM reflectivity detection of nanoparticles. Also incorporated herein by reference in its entirety is U.S. patent application Ser. No. 09/969,518, “Method of Manufacture of Colloidal Rod Particles as Nanobarcodes,” filed Oct. 2, 2001, which discloses photolithographic methods for manufacturing the rod-shaped nanoparticles.
Similar structures have been formed as nanowires for use in electronic applications. For example, nanowire diodes have been synthesized by sequential electroplating of metals and assembly of semiconductor/polymer films (N. I. Kovtyukhova et al., “Layer-by-Layer Assembly of Rectifying Junctions in and on Metal Nanowires,” J. Phys. Chem. B 2001, 105, 8762-8769). These diodes consist of 10-nm thick semiconductor/polymer films sandwiched between two 0.5-300 μm segments of a 200-nm diameter nanowire. These particles are too large to be SERS active. Much smaller 30-nm diameter nanowires have also been produced containing alternating 5-nm thick Ni and Cu layers (L. Sun et al., “Fabrication of Nanoporous Single Crystal Mica Templates for Electrochemical Deposition of Nanowire Arrays,” J Mater. Sci. 2000, 35, 1079). These nanowires are not SERS-active and are not used as biomolecular or other tags.
Given the dramatic enhancement of SERS activity observed when a Raman-active molecule is sandwiched between two SERS-active substrates, it is desirable to have a method for deliberately preparing such sandwiches. Particles prepared with such methods would have utility as optical tags.